The applications of lactic acid and its derivatives encompass many fields of industrial activities (i.e., chemistry, cosmetic, and pharmacy), as well as important aspects of food manufacture and use. Furthermore, today there is growing interest in the production of such an organic acid to be used directly for the synthesis of biodegradable polymer materials.
Lactic acid may be produced by chemical synthesis or by fermentation of carbohydrates using microorganisms. The latter method is now commercially preferred because microorganisms have been developed that produce exclusively one isomer, as opposed to the racemic mixture generated by chemical synthesis. The most important industrial microorganisms, such as species of the genera Lactobacillus, Bacillus, and Rhizopus, produce L(+)-lactic acid. Production by fermentation of D(−)-lactic acid or mixtures of L(+)- and D(−)-lactic acid are also known.
During a typical lactic acid fermentation, there is an inhibitory effect caused by lactic acid produced on the metabolic activities of the producing microorganism. Besides the presence of lactic acid, lowering the pH value also inhibits cell growth and metabolic activity. As a result, the extent of lactic acid production is greatly reduced.
Therefore, the addition of Ca(OH)2, CaCO3, NaOH, or NH4OH to neutralise the lactic acid and to thereby prevent the pH decrease is a conventional operation in industrial processes to counteract the negative effects of free lactic acid accumulation.
These processes allow the production of lactate(s) by maintaining the pH at a constant value in the range of about 5 to 7; this is well above the pKa of lactic acid, 3.86.
Major disadvantages are connected to the neutralisation of lactic acid during the fermentation. Mainly, additional operations are required to regenerate free lactic acid from its salt and to dispose of or recycle the neutralising cation; this is an expensive process. All the extra operations and expense could be eliminated if free lactic acid could be accumulated by microorganisms growing at low pH values, thus minimising the production of lactate(s).
It has been proposed the use of recombinant yeasts expressing the lactate dehydrogenase gene so as to shift the glycolytic flux towards the production of lactic acid.
FR-A-2 692 591 (Institut Nationale la Recherche Agronomique) discloses yeast strains, particularly Saccharomyces strains, containing at least one copy of a gene coding for a lactate dehydrogenase from a lactic bacterium, said gene being under the control of sequences regulating its expression in yeasts.
Said strains may give both the alcoholic and the lactic fermentation and this so called “intermediate” or “balanced” fermentation could be exploited in areas such as brewing, enology, and baking.
Porro et al., (Biotechnol. Prog. 11, 294-298, 1995) have also reported the transformation of S. cerevisiae with a gene coding for bovine lactate dehydrogenase.
However, because of the high production of ethanol, the yield in the production of lactic acid for both the processes described was not considered to be competitive with that obtainable by the use of lactic bacteria.
In the past decade, “non conventional yeasts” other than S. cerevisiae have gained considerable industrial interest as host for the expression of heterologous proteins. Examples are the methanol-utilising yeasts such as Hansenula polimorpha and Pichia Pastoris, the lactose-utilizing yeasts such as Kluyveromyces lactis. In addition to enabling the use of a wider range of substrates as carbon and energy sources, other arguments have been put forward to the industrial use of “non conventional yeasts”. Generally speaking, biomass and product-yield are less affected, in some of these yeasts, by extreme conditions of the cellular environment. High-sugar-tolerant (i.e., 50-80% w/v glucose medium; Torulaspora-syn. Zygosaccharomyces-delbrueckii, Zygosaccharomyces rouxii and Zygosaccharomyces bailii; Ok T and Hashinaga F., Journal of General & Applied Microbiology 43(1): 39-47, 1997) and acid- and lactic-tolerant (Zygosaccharomyces rouxii and Zygosaccharomyces bailii; Houtsma P C, et al., Journal of Food Protection 59(12), 1300-1304, 1996.) “non conventional yeasts” are available. As already underlined the cost of down stream processing could be strongly reduced if the fermentation process is carried out under one or more of the above mentioned “extreme conditions”.